Expression of pcp4a in Subpopulationsof CNS Neurons in Zebrafish

MARINA MIONE,1,2* ZSOLT LELE,1,3 CAMILLA T. KWONG,1 MIGUEL L. CONCHA,4

AND JONATHAN D. CLARKE1

1Department of Anatomy and Developmental Biology, University College London,London WC1E 6BT, United Kingdom

2Istituto Fondazione Italiana Per La Ricerca Sul Cancro (FIRC) Di Oncologia Molecolare,FIRC Institute of Molecular Oncology, 20139 Milano, Italy

3Institute of Experimental Medicine, H-1083 Budapest, Hungary4Anatomy and Developmental Biology Program, Faculty of Medicine, Institute of

Biomedical Sciences, Universidad de Chile, Santiago Clasificador 7–Correo 7, Chile

ABSTRACTThe molecular organization of the zebrafish brain and its relation to neuroanatomical

divisions are still largely unknown. In this study we have analyzed the expression of a smalltranscript encoding for the IQ containing polypeptide Pcp4a in developing and juvenilezebrafish. The transcript is exclusively expressed in neural structures with a pattern that ishighly specific for restricted domains and cell populations throughout development, and itallows us to follow the development of these structures at different times. The expression ofpcp4a characterizes the dorsocaudal telencephalon, dorsal habenula, pretectal nuclei, pre-glomerular complex, mammillary bodies, and deep layers of the optic tectum and is ahallmark of a subpopulation of reticulospinal neurons. In the telencephalon, comparison ofthe expression of pcp4a with other pallial markers showed a rostrocaudal gradient in theexpression of these genes, which suggests that the dorsal telencephalon of zebrafish may beorganized in distinct areas with different molecular natures. Pcp4 has been involved inmodulating calcium signals and in binding to calmodulin, but its precise role in neuronalfunctions is not known. The analysis of pcp4a expression and localization in the zebrafishbrain suggests that pcp4a may be a useful marker for sensory and some motor neuronalcircuitries and for telencephalic areas processing sensory inputs. J. Comp. Neurol. 495:769–787, 2006. © 2006 Wiley-Liss, Inc.

Indexing terms: telencephalon; teleost; tbr1; reelin; pattern

The zebrafish has become the leading teleost model forstudies of molecular genetics and embryology. Forward ge-netics through mutagenesis screens has yielded a large num-ber of mutations in many developmental processes, and nodoubt the list of mutated genes will soon be extended togenes affecting behavior or complex neuronal functions.However, knowledge of the organization, connectivity, andfunctions of zebrafish brain areas is disappointingly scanty.An understanding of how the zebrafish brain is patternedand wired and the molecular nature of its divisions will helpto address issues of structure-function relationship.

One possible approach is to use molecular markers thatin other species have proved helpful in identifying themajor divisions of the vertebrate brain during develop-ment (Puelles et al., 2000; Bachy et al., 2002; Brox et al.,2004; Medina et al., 2004). The rationale behind this is theunderstanding that regulatory genes consistently ex-pressed in the same or a homologous domain in different

species play a fundamental role in the development of thespecialized features of that area.

In parallel to the analysis of regulatory gene expressiondomains in the developing zebrafish brain, we have

Grant sponsor: the Wellcome Trust (to S.W.W., M.L.C.); Fondo Nacionalde Desarrollo Cientıfico y Tecnologico; Grant number: 1020902; Grantnumber: 7020902 (to M.L.C.); Grant sponsor: Iniciativa Cientifica Milenio;Grant number: P04-068-F (to M.L.C.); Grant sponsor: Programa Bicente-nario Ciencia y Tecnologia (to M.L.C.); Grant number: ACT 47.

*Correspondence to: Marina Mione, Ifom, FIRC Institute of MolecularOncology, Via Adamello, 16, 20139 Milano, Italy.E-mail: marina.mione@ifom-ieo-campus.it

This article includes Supplementary Material available at http://www.interscience.wiley.com/jpages/0021- 9967/suppmat

Received 17 July 2005; Revised 9 October 2005; Accepted 16 November2005

DOI 10.1002/cne.20907Published online in Wiley InterScience (www.interscience.wiley.com).

THE JOURNAL OF COMPARATIVE NEUROLOGY 495:769–787 (2006)

© 2006 WILEY-LISS, INC.

started a systematic search for transcripts specifically ex-pressed during development, using both bioinformatic ap-proaches and classical subtractive hybridization tech-niques. Those transcripts that show a specific pattern ofexpression and, based on sequence analysis, encode forfunctionally relevant products are used for detailed anal-ysis of their expression. We hope that this approach willgive insights into the functional divisions of the zebrafishbrain during development and complement the molecularand neuroanatomical data. Moreover, the identification oftranscripts with specific patterns of expression representsan indispensable tool aiding in the identification of mu-tated genes causing neuronal specific phenotypes.

Purkinje cell peptide 4 (Pcp4), also known as Pep19 (Ziaiet al., 1986), is a small polypeptide carrying an IQ motif: a23-amino acid domain that binds EF-hand proteins andshows high affinity for calcium-poor calmodulin (Ziai et al.,1986; Skene, 1989; Liu and Storm, 1990; Johanson et al.,2000; Bahler and Rhoads, 2002). The expression of pcp4 insubpopulations of mature neurons of mouse and rat centralnervous system (CNS) has been correlated with their abilityto undergo long-term potentiation (LTP) and long-term de-pression (LTD) and their resistance to apoptotic insults (Jo-hanson et al., 2000). Other members of the IQ motif class ofpolypeptides, besides Pcp4, are neuromodulin or GAP-43and neurogranin or RC3, all neuron-specific peptides (Slem-mon et al., 2000) expressed both during development and inmature neurons.

Recently, the expression patterns of Pcp4 (Bulfone et al.,2004; Thomas et al., 2003) and those of a related peptide,Pcp4-like1 (pcp4L1; Bulfone et al., 2004) have been reportedin the developing mouse embryo, demonstrating that the

expression of these genes is not confined to mature CNSstructures. We isolated a cDNA clone (pcp4a) encoding for azebrafish pcp4 polypeptide through the screening of an adultzebrafish brain library with a complex probe representingtranscripts preferentially expressed in the embryonic brain.A second transcript (pcp4b) encoding for a highly similarpeptide and one encoding for a Pcp4-like1 peptide were iden-tified through a BLAST search of the expressed sequence tag(EST) database at the National Center for BiotechnologyInformation (NCBI). Their expression patterns differslightly from that of pcp4a and will be reported separately.The identification of the transcript for pcp4a in an adultbrain library screened with a probe enriched for developmen-tally regulated transcripts indicated that the gene was ex-pressed throughout development, and the analysis of itsexpression pattern suggested a highly specific expression inneuronal groups.

Here we report the expression of pcp4a throughout de-velopment: a comparison with the expression of othergenes marking the same or adjacent population of neuronsallows us to consider pcp4a as a novel marker for cellgroups that so far are not known to be specifically markedby other gene expression. Moreover, the analysis of pcp4aexpression in the dorsal telencephalon suggests the exis-tence of areal divisions that may be related to the func-tional organization of the telencephalon in zebrafish.

MATERIALS AND METHODS

Cloning of the zebrafish pcp4a gene

Hybridization of a microarrayed adult brain zebrafishlibrary distributed by RZPD (Berlin, Germany; library no.

Abbreviations

AC anterior commissureCC crista cerebellarisCce cerebellar bodyCer cerebellumCM corpus mammillare, mammillary bodyCP central posterior thalamic nucleusCT caudal telencephalonD dorsal telencephalic areaDc central zone of DDd dorsal zone of DDH dorsal hornDIL diffuse nucleus of inferior lobeDl lateral zone of DDm medial zone of DDp posterior zone of DECL external cellular layer of olfactory bulb including mitral

cellsEn entopeduncular nucleusep epiphysisET eminentia thalamigc central grayGCL ganglion cell layerGL glomerular layer of the olfactory bulbHa habenulaHad dorsal habenular nucleusHadl dorsal habenular nucleus lateral regionHadm dorsal habenular nucleus medial regionHav ventral habenular nucleusICL internal cellular layer of the olfactory bulbIO inferior oliveIL intermedio-lateral columnLFB lateral forebrain bundlelot lateral olfactory tractLVII facial lobeLX vagal lobe

mhb mid-hindbrain boundarymot medial olfactory tractnMLF nucleus of medial longitudinal fascicleON octaval nucleiOT optic tectumOtr optic tractOs optic stalkov otic capsulePGC preglomerular nuclear complexPGZ periventricular gray zone of the optic tectumPoa preoptic areaPPp parvocellular preoptic nucleusPTN pretectal nucleiRF reticular formationRS reticulospinal neuronSC suprachiasmatic nucleusSD saccus dorsalisSG nucleus subglomerulosusSGN secondary gustatory nucleusTL torus longitudinalisTLA lateral torusTPM tractus pretectomammillarisTPp periventricular nucleus of posterior tuberculumTS torus semicircularisV ventral telencephalic areaVA valvulaVd dorsal nucleus of VVH ventral hornVl lateral nucleus of VVL ventrolateral thalamic nucleusVM ventromedial thalamic nucleusVs supracommissural nucleus of VVT ventral thalamusVv ventral nucleus of V

770 M. MIONE ET AL.

Fig. 1. Alignment of pcp4 sequences. A: Optimal alignment ofamino acid sequences of human, mouse, rat, pig, cow, chick, Xenopus,and zebrafish Pcp4 sequences was obtained with the Clustal-X pro-gram and checked manually. The consensus amino acid sequence is atthe top of each group, and the IQ motif is underlined at the bottom.

B: A distance tree of the Pcp4 proteins isolated from different verte-brate species. The distance tree was drawn with the Neighbor-joiningprogram from the Phylip package. As the tree indicates, zebrafish has2 pcp4 genes (named pcp4a and pcp4b) and only one pcp4L1 gene.Numbers indicate the bootstrap values.

Fig. 2. pcp4a expression in the brain of 24-hpf zebrafish. A–H:Lateral (A,E), dorsal (B–D,F,H), and anterior (G) views of pcp4aexpression in whole embryos/brains. A,C: Double staining with pax2.1and pcp4a probes (colors as indicated in the picture). Arrowheadspoint to pcp4a expression in the RS neuron precursors. B: Doublestaining with krox20 and pcp4a probes. Arabic numbers indicaterhombomeres. D: Double staining with val and pcp4a probes. Arrowindicates double staining in RS cells in rhombomere 5. E: Expressionof pcp4a in the epiphysis. E� Inset shows a dorsal view of the dien-cephalon with the expression of pcp4a at the two lateral poles of theepiphysis. E��: Inset shows pcp4a expression (blue) in two RS neurons(Mid3cm) retrogradely labeled with biotin dextran (brown). F: Double

staining with islet1 and pcp4a probes. Black arrows point to pcp4a-expressing RS neuron precursors; white arrowhead points to thefacial motor nucleus, and white arrow indicates the octaval nucleus,both expressing islet1. G: Double staining with GFP antibody and apcp4a probe in an embryo of the tg(foxD3-GFP) line. Arrows point topcp4a-expressing cells and their axons. H: pcp4a expression in thespinal cord at 3 dpf. I: Double staining with GFP antibody (brown)and a pcp4a probe (blue) in an embryo of the tg(islet1-GFP) line.Arrowheads point to double-stained neurons. For abbreviations, seelist. Scale bars � 100 �m in A–I. [Color figure can be viewed in theonline issue, which is available at www.interscience.wiley.com.]

772 M. MIONE ET AL.

611) with a complex probe generated through subtractivehybridization (Mione, unpublished data) yielded a numberof positive clones, which were analyzed for their sequenceand expression pattern. The complex probe was generatedwith the ClonTech (Palo Alto, CA) PCR Select Kit, whichis based on the RDA method (Lisitsyn et al., 1993), follow-ing subtraction of mRNAs common to adult zebrafishbrain and 24-hpf whole zebrafish embryos. The remainingmRNAs (representing genes expressed predominantly indeveloping embryos) were used to synthesize a cDNAprobe in the presence of 32P-dCTP. With this complex(so-called as it represents several mRNAs) probe wescreened an arrayed cDNA library generated from adultzebrafish brain by John Ngai (University of California atBerkeley, CA) and distributed by RZPD (www.rzpd.de).The goal of this strategy was double: 1) to identify full-length clones, corresponding to the 300-nt polymerasechain reaction (PCR) fragments generated by the subtrac-tion approach, in order to facilitate the identification of

the clones through sequencing; and 2) to restrict furtherwork to clones expressed exclusively in the CNS.

In situ hybridization for two of these clones with iden-tical sequence gave a neuronal specific pattern. Concep-tual translation yielded an open reading frame of 62amino acids, highly similar to that of mouse and humanPcp4.

Fish strains

Danio rerio of wild-type and transgenic lines were fromthe UCL Zebrafish Facility. The tg(islet-1-GFP) line wasdonated by H. Okamoto (Higashijima et al., 2000). Thetg(foxD3-GFP) line was donated by C. Nusslein-Volhard(Gilmour et al., 2002). Backfilling experiments were car-ried out according to European Experimental AnimalCare guidelines.

In situ hybridization

Antisense probes for pcp4a, reelin (Costagli et al., 2002),and tbr1 (Mione et al., 2001) were generated by lineariza-tion with SalI and in vitro transcription with SP6 in thepresence of digoxigenin (dig)-labeled ribonucleotides(Roche, Indianapolis, IN). Other plasmids used in thisstudy were: pax2.1 (Macdonald et al., 1997); krox20 (Ox-toby and Jowett, 1993); islet1 (Korzh et al., 1993); val(Moens et al., 1996); emx1, emx2, and emx3 (Morita et al.,1995); and pax6.1 and -6.2 (Nornes et al., 1998). Singleand double in situ hybridization with two color substrateswas performed according to Hauptmann and colleagues(2002). Substrates used in this study are 5-bromo-4-chloro-3-inodoyl phosphate (BCIP)/NBT for blue color andINT/BCIP for red color, both from Roche.

In situ hybridization of cryostat sections, followed or notby immunocytochemistry for green fluorescent protein(GFP), was performed as previously described (Costagli etal., 2002).

Backfilling

We performed three types of backfilling experiments.First, to compare the pattern of expression of pcp4a withthe localization of reticulospinal neurons in wholemountpreparations of the hindbrain, we injected fluorescein dex-tran (molecular weight 10,000; Molecular Probes, Eugene,OR) into the upper spinal cord of 4-dpf zebrafish as de-scribed (Alexandre et al., 1996) to label the reticulospinal(RS) neurons retrogradely. Survival time after this injec-tion was 12–18 hours. Fluorescein dextran was revealedwith a dig-labeled anti-fluorescein antiserum (Roche) usedat a concentration of 1:10,000 in paraformaldehyde-fixedlarvae and revealed by using BCIP/NBT as the substrate,as described previously (Costagli et al., 2002). In a secondset of backfilling experiments, designed to reveal expres-sion of pcp4a in backfilled RS neurons, we used biotinyl-ated dextran as the tracer (Molecular Probes) and thesame labeling/survival parameters used in the previousbackfilling experiment. To reveal the co-existence of pcp4atranscripts and biotin dextran, we sectioned the hind-brains of injected larvae with a cryostat and subjected theserial sections to in situ hybridization for pcp4a, as de-scribed above. Following the detection of the pcp4a tran-scripts with BCIP/NBT as the substrate, we incubated thesections with a streptavidin-horseradish peroxidase com-plex (ABC, Vector, Burlingame, CA) following the manu-facturer’s instructions and revealed the biotinylated dex-tran with a diaminobenzidine (DAB) and H2O2 substrate

Fig. 3. pcp4a expression at 48 hpf. Lateral (A), dorsal (B), andventral (C) views of 2-dpf zebrafish stained with a pcp4a probe. Forabbreviations, see list. Scale bar � 50 �m in C (applies to A–C).

773pcp4a IN ZEBRAFISH NEURONS

Fig. 4. Distribution of pcp4a mRNA transcripts. Drawings of aseries of transverse sections through the zebrafish brain, depictingthe distribution of pcp4a transcripts. A: Schematic drawing of a

lateral view of an adult zebrafish brain, to show the levels and orien-tation of the sections in B–M. pcp4a transcripts are represented byblack dots on the right side of the sections. For abbreviations, see list.

774 M. MIONE ET AL.

as described in the double in situ/immunocytochemistryprotocol (Costagli et al., 2002).

A third set of backfilling experiments was per-formed to analyze spinal cord intersegmental and longprojection neurons and their relation to pcp4a expres-sion. We labeled subpopulations of spinal cord neuronswith intraspinal injections (at the level of the yolk ex-tension) of a mixture of biotinylated dextran and fluo-rescein dextran at 5 dpf as described (Higashijima et al.,

2004). The day after, larvae were examined for thedistribution of fluorescent labeling and fixed with 4%paraformaldehyde (PFA; in phosphate-buffered saline[PBS], pH 7.4). Labeled regions of spinal cords wereprocessed for pcp4a in situ hybridization on cryostatsections as described above, reacted with horseradishperoxidase (HRP)-conjugated streptavidin (VectorABC), and revealed with DAB and H2O2 substrate in-cubation.

Figure 4 (Continued)

775pcp4a IN ZEBRAFISH NEURONS

Figure 5.

Photography

Images were captured with a Jenopick digital camera(Improvision, Coventry, UK) connected to a Nikon Eclipse1000 microscope, using �4, �20, and �40 Plan-apo lensesand Open Lab software (Improvision). Digital images werestored as 1,600 � 1,200 pixels at a resolution of 300 dpiand manually arranged to form composite pictures byusing Adobe Photoshop 7 (San Jose, CA).

RESULTS

During a screening of an adult zebrafish brain cDNAlibrary with a complex probe representing transcriptspreferentially expressed in embryonic brains, two identi-cal clones were identified, which represented the samefull-length cDNA encoding for a putative pcp4 peptide(pcp4a; Fig. 1A, accession number: DQ122930). A searchof zebrafish and Xenopus databases revealed several ESTclones that, when conceptionally translated, gave the se-quences reported in Figure 1A. The zebrafish EST se-quences corresponded to two different transcripts, oneidentical to pcp4a and another slightly divergent, whichwe called pcp4b. Differences in the sequences suggestedthat the two transcripts were those of two different genesthat may have resulted from the well-documented teleost-specific gene duplication event that took place about 350–400 million years ago (Amores et al., 1998), rather thanalternatively spliced transcripts of the same gene. This isfurther confirmed by the different chromosomal location oftwo Ensemble transcripts corresponding to pcp4a andpcp4b (data not shown). Thus pcp4a and pcp4b are verylikely to be paralogues. A third class of transcripts encod-ing for another IQ-containing peptide highly similar topcp4a and -b was identified through a Blast search. Thesetranscripts encode for the zebrafish orthologue of pcp4L1(Bulfone et al., 2004), as suggested by phylogenetic anal-ysis of sequences from different species (Fig. 1B).

High similarity is found among all vertebrate se-quences, with the Xenopus clone being the most divergent(Fig. 1B). The IQ motif was identified in all sequences(underlined in Fig. 1A).

Expression of pcp4a during embryonicdevelopment

The expression of pcp4a in zebrafish starts around 22hpf (26-somite stage) in two bilateral cellular groups lo-cated in the lateral region of the hindbrain (Fig. 2A,B).

Double labeling with a pax2.1 probe that marks the oticcapsule shows that pcp4a-expressing cells are located me-dial to it (Fig. 2A,C). Double labeling with a krox20 cRNAprobe at 22 hpf to mark rhombomeres 3 and 5 shows thatpcp4a-expressing cells are located at the level of rhom-bomere 5 and 6 (Fig. 2B). To gain insights into the identityof these groups of cells expressing pcp4a we performeddouble in situ hybridization with a val probe, which labelsMauthner neurons and other reticulospinal neurons(Moens et al., 1996). The paired group of cells expressingpcp4a located in rhombomere 5 co-expresses val, suggest-ing that they maybe reticulospinal neurons (Fig. 2D). Thishas been shown by double labeling of reticulospinal neu-rons using retrograde back filling and pcp4a in situ hy-bridization at 5 dpf (Figs. 2E��, 8).

By 30 hpf the presumptive reticulospinal neurons la-beled by the expression of pcp4a are located in a moremedial position. Double labeling with a islet-1 probe tolabel cranial nerve nuclei (Fig. 2F) shows that the uppergroup of pcp4a-expressing cells in the hindbrain is next toneurons of the octaval nerve nuclear complex, whereas thelower group is located just lateral to neurons of the facial(VII) nerve nucleus. Another domain of expression ap-pears in the epithalamus at 30 hpf, where pcp4a markstwo groups of cells in the lateral poles of the epiphysis(Fig. 2E,E�). Analysis of pcp4a expression at 48 hpf in atransgenic line expressing GFP specifically in pineal neu-rons and their projections, tg(foxD3-GFP), allowed us toidentify the pineal cells expressing pcp4a tentatively asprojection neurons, because they give rise to GFP� axonsprojecting out of the epiphysis into the lateral forebrainbundle (Fig. 2G and data not shown). Expression of pcp4ais also detected in a chain of dorsal spinal cord neurons,some of which are GFP� in the tg(islet1-GFP) line, sug-gesting that they may be Rohon Beard neurons (Fig. 2H).However, not all cells express both markers (Fig. 2I).

At 48 hpf, analysis of pcp4a expression reveals addi-tional domains (Fig. 3A–C). These include the caudal tel-encephalon, the preoptic area, the pretectal nuclei, severalnuclei in the mesencephalic tegmental area, restrictedcells in the optic tectum, and cells at the mid-hindbrainboundary, in the hindbrain, and throughout the ventralregion of the spinal cord, plus cells in the central portion ofthe ganglion cell layer in the retina.

Thus, by 48 hpf, expression of pcp4a has spread to alarge number of neuronal cell populations likely to havejust become functional active.

Expression of pcp4a at 4 days, 2 weeks, and1 month post fertilization

The expression of pcp4a was analyzed at 4–5 dpf, 2weeks, and 1 month post fertilization by using cryostatsections to identify the areas and cell groups that expresspcp4a during development. The pattern of expression ofpcp4a is similar at the three age end-points, with a fewexceptions (i.e., in the retina); therefore the description ofthe expression pattern of pcp4a will be carried out inparallel for all the age points, and examples of the stainingat the different ages will be provided. Because morpho-genesis of many brain structures is still ongoing between4 dpf and 1 month, the comparison of pcp4a expressionbetween these two stages reveals how morphogeneticmovements and restricted proliferation influence the loca-tion and size of brain nuclei expressing pcp4a. An over-view of the expression pattern of pcp4a in the brain of

Fig. 5. pcp4a expression in the telencephalon and diencephalon.A,C,E: Sagittal sections through the brain of 2-week (A,E), and1-month-old (C) zebrafish processed to reveal the distribution of pcp4atranscripts. The pattern of pcp4a expression at the two stages issimilar, but the levels of the transcripts are reduced at 1 month.B,D,F,G: Transverse sections of 2-week (B,F) and 1-month-old (D,G)zebrafish. A:: Forebrain region. The black line indicates the level ofthe transverse section in B. The box indicates the enlarged area in E.B:: Telencephalic region just caudal to the anterior commissure. Ar-rows point to skin melanocytes. C: Forebrain region, cfr with A. Theblack line indicates the level of the transverse section in D. D: Telen-cephalon, caudal to the anterior commissure. Compare the reducedlevels of pcp4a transcripts with B. E: Enlargements of the epithalamicregion shown in A. F,G: Transverse sections (and schematic drawing,H) of the epithalamic expression of pcp4a at 2 weeks and 1 month. Forabbreviations, see list. Scale bars � 50 �m in A–G.

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Fig. 6. pcp4a expression in the diencephalon and mesencephalon.A: Sagittal and (B–E) transverse sections showing pcp4a expressionin the mesencephalon at 1 month (A,E,F), 2 weeks (C), and 4 dpf (B).

Black lines with letters in A indicate the levels of the correspondingtransverse sections. F: Section through the trigeminal ganglion at 1month. For abbreviations, see list. Scale bars � 70 �m in A–F.

778 M. MIONE ET AL.

Figure 7.

1-month-old zebrafish is presented schematically in Fig-ure 4.

pcp4a expression in the telencephalon. At 5 dpf and2 weeks, pcp4a transcripts were detected in the laterome-dial region of the dorsal telencephalon (specifically in theDl and Dp; Fig. 5A,B). However, by 1 month, pcp4a isexpressed mainly in the caudal third of the telencephalonin both the dorsal and ventral telencephalon (Fig. 5C,D).In the ventral telencephalon, pcp4a is expressed in theentopeduncular nucleus (En; Fig. 5A,E) and in the parvo-cellular preoptic nucleus (PPn; Fig. 5D) caudal to theanterior commissure (Fig. 5A). There is no expression inthe olfactory bulb.

pcp4a expression in the diencephalon. In the dien-cephalon, pcp4a is strongly expressed in the dorsal habe-nular nucleus (stronger in the lateral than in the medialportion; Fig. 5E,F), in the presumptive eminentia thalami(ET; Fig. 5E), in the migrated pretectal nuclear complex(only present at 1 month; Fig. 5G,H; present in both theirperiventricular and migrated groups at 5 dpf, Fig. 6A,B),and in the central nucleus of the dorsal thalamus (CP; Fig.6C,D). Low levels of expression of pcp4a were also de-tected in the nuclei of the posterior tuberculum (TPp; Fig.6D). In addition, large cells in the nucleus of the mediallongitudinal fascicle (nMLF) express pcp4a (Fig. 6E), andfinally, the nucleus subglomerulosus (SG) and the toruslateralis (TLA) are among of the most intensely labeledareas (Fig. 6A,E). The preglomerular complex (PGC) ex-presses the highest levels of pcp4a transcripts in the di-encephalon (Fig. 6A,C,D).

pcp4a expression in the hypothalamic region. Be-sides a faint expression of pcp4a in the anterior periven-tricular region (Fig. 6C), the caudal hypothalamus ex-presses the highest levels of pcp4a in its pairedmammillary bodies (Fig. 7A,C); a low expression of pcp4ais also present in the diffuse nucleus of the inferior lobe(DIL; Fig. 6E). Note that at 5 dpf, the mammillary bodiesare located in a lateral position in the hypothalamic anla-gen (Fig. 7B, arrowheads) whereas at 1 month they occupythe medioventral region of the caudal hypothalamus (Fig.7C).

pcp4a expression in the mesencephalon. Expressionof pcp4a can be detected in the periventricular gray zone(PGZ) of the optic tectum and in sparse cells of the stra-tum griseum centrale (Figs. 6A–F, 7A,B). Large cells atthe ventral side of the torus longitudinalis (TL) expresspcp4a, throughout its rostrocaudal extension (Fig. 7D,E),whereas the majority of the granule-like cells of TL ex-press reelin (cf. Fig. 4b in Costagli et al., 2002). In the

torus semicircularis (TS), two groups of cells expresspcp4a transcripts (Figs. 6A,E, 7A,B). One group is com-posed of medium cells found in the lateral torus semicir-cularis, close to the tectal ventricle. The other group con-sists of large cells in the ventral part (layer 1, Ito, 1974).At a more caudal level, the secondary gustatory nucleus(SGN; Wullimann et al., 1996) expresses low levels ofpcp4a (not shown).

pcp4a expression in the rhombencephalon and spi-

nal cord. Expression of pcp4a can be detected at lowlevels in the Purkinje cells of the valvula and crista cer-ebelli (Fig. 7D–F) but not in the corpus cerebelli. A similarrelation between reelin� granule cells and pcp4a� largeneurons is found in the TL (see above), valvula, and cristacerebellaris (Fig. 7D,E), suggesting that these structuresmay share similar molecular identities. Note that thePurkinje cells of the corpus cerebelli do not express pcp4a(Fig. 7D, asterisks). Cells in deep layers of lobus VII and inthe superficial layer of lobus X (sensory relay nuclei for thefacial and vagal nerves, respectively) express pcp4a (Fig.8D).

pcp4a continues to be expressed by reticulospinal neu-rons at 5 days (Fig. 8B,C,G) and 1 month post fertilization,when most, but not all, reticulospinal neurons labeled bybackfilling with fluorescein dextran (Fig. 8A) appear toexpress pcp4a (Fig. 8B,C). Notably, Mauthner cells do notexpress pcp4a (Fig. 8A–C,J), whereas their homologues inr5, Mid3cm, do express pcp4a throughout all developmen-tal stages studied (Figs. 2, 8C,G,H,K). pcp4a transcriptscould also be detected in the descending axons of somereticulospinal neurons (Fig. 8E, arrow).

In the spinal cord, neurons of different sizes, includingvisceral motor neurons expressing reelin (Costagli et al.,2002), express pcp4a throughout the length of the spinalcord (Fig. 9B,E). For visceral motor neurons and primarysensory neurons in the dorsal root ganglia (which do notexpress reelin; Fig. 9A,C, arrow), compare reelin andpcp4a expression in adjacent sections (Fig. 9A,C). Besidesthe visceral motor neurons, a large number and variety ofspinal cord cells express pcp4a. In order to assess whetherthe cells that express pcp4a in the spinal cord includemotor neurons or interneurons, we performed intraspinalinjections of biotinylated dextran to mark the interneuronpopulation and assessed the expression of pcp4a in thelabeled cells. Most classes of interneurons, including theCiD, CoLA, CoBL, and UCoD neurons (as defined by Haleet al., 2001 and Higashijima et al., 2004) labeled at 5 dpfby this procedure express pcp4a (Fig. 9E–G).

pcp4a expression in the retina and trigeminal sen-

sory ganglion. From 48 hpf pcp4a is expressed in theganglion cell layer of the developing retina (Fig. 3). Theexpression is initially confined to the medial sector andthen spreads to the rest of the ganglion cell layer by 4 dpf(Fig. 6B). Sparse cells in the inner nuclear layer alsoexpress pcp4a (Fig. 6B). No expression of pcp4 is detect-able in the retina of 1-month-old zebrafish (data notshown). By 4 dpf, expression of pcp4 is also found in a fewlarge neurons of the trigeminal sensory ganglion; thisexpression is still detectable at 1 month (Fig. 6E, arrow-head, Fig. 6F).

Comparison of the expression domains of tbr1, ree-

lin, and pcp4 in the telencephalon. In mammals, pcp4is strongly expressed in the dorsal telencephalon at lateembryonic stages through adulthood, and its transcriptslocalize predominantly to the hippocampal region

Fig. 7 (Overleaf). pcp4a expression in the hypothalamus, mesen-cephalon and rhombencephalon. Sagittal (A,D–F) and transverse(B,C) sections showing pcp4a expression at 1 month (A,C), 2 weeks(D–F), and 5 dpf (B) in the mesencephalic and rhomboencephalicregions of the zebrafish brain. A: Black line indicates the level of thetransverse section shown in C. B: Arrowheads point to the mammil-lary bodies, which at 5 dpf are located in a more dorsal and lateralposition than at 2–4 weeks. C: Arrows indicate the intensely stainedmammillary bodies. D: Sagittal section through the isthmic region.Asterisks indicate the unstained corpus cerebelli. Boxed areas areenlarged in E and F. E,F: In E, arrowheads point to large cells in thetorus lateralis, and arrow indicates large cells in the valvula. In F,arrow points to large cells in the crista cerebellaris. For abbreviations,see list. Scale bars � 50 �m in A–F.

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Figure 8.

(Thomas et al., 2003). Thus zebrafish pcp4a is a poten-tially useful marker for this region, which has not yet beenproperly identified in teleosts. Therefore we searched forother telencephalic markers that are expressed in devel-oping, but also mature, zebrafish brain, to compare theirdistribution with that of pcp4a. We chose tbr1 and reelinfor multiple reasons (see Discussion) but mainly becausethey are expressed in the dorsal telencephalon from de-velopmental to adult stages. In addition, analysis of othermarkers (namely, emx and pax6 genes) showed that thesegenes (with the exception of emx3; see supplementary Fig.1S) are not expressed in the dorsal telencephalon of 5-dpfzebrafish. In the brain of 5-dpf zebrafish, expression ofreelin is strong at the rostral pole of the dorsal telenceph-alon at the border with the olfactory bulb and in themedial regions of the dorsal telencephalon (Fig. 10A,B),whereas it is absent in the lateral regions, especially inthe caudal telencephalon (arrowhead, Fig. 10B).

By contrast, tbr1 is strongly expressed in cells of thedorsal and laterodorsal telencephalon, particularly in thecaudal telencephalon, where its expression domain contin-ues ventrally into the diencephalic eminentia thalami/lateral forebrain bundle region (asterisk in Fig. 10C,D)through the entopeduncular nucleus (arrow, Fig. 10C).The pcp4a expression domain in the dorsal telencephalonoverlaps with both reelin and tbr1 (Fig. 10E,F). However,unlike tbr1 and similarly to reelin, it is strongly expressedin the medial regions of the dorsal telencephalon (Fig.10E,F). Another difference between tbr1 and pcp4a ex-pression in the forebrain is the presence of pcp4a, but nottbr1, transcripts in the habenular nuclei (arrowhead, Fig.10F). Thus the combined expression of these three genesidentifies at least three distinct areas of the dorsal pal-lium, a region of the dorsal telencephalon that in mam-mals evolved to form the isocortex, in developing zebrafishlarvae: an anteromedial dorsal pallial area, which showsstrong expression of reelin and pcp4a, a caudolateraldorsal-pallial area that shows strong expression of tbr1and pcp4a, and a dorsal-pallial area proper that showsstrong expression of all three of these genes (Fig. 10G,H).Outside the dorsal pallial regions, the expression of thethree genes shows other interesting overlaps: tbr1 and

pcp4 overlap in the ventralmost part of the Dm, posteriorto the anterior commissure (white arrows in Fig. 10A,C,E;white areas in Fig. 10G,H); tbr1 is expressed alone in thelateralmost part of the Dp (black arrow in Fig. 10C; redarea in Fig. 10G).

DISCUSSION

Our study of the distribution of the transcripts of thesmall peptide pcp4a in brain areas and cell groups duringzebrafish development identifies a new marker for specificneuronal populations through morphogenesis and migra-tion until adulthood. Another outcome of this study is thatthe expression domains of pcp4a in the zebrafish brain arelargely similar to those observed in the developing mousebrain for an orthologue gene, pcp4, thus allowing us toconsider the expression of this peptide as a marker ofbrain areas or neuronal populations in two different ver-tebrates whose lineages diverged more than 400 millionyears ago (Carroll, 1988).

As a result of the observation that pcp4a expressionhighlights conserved domains in the fish and mammalianbrain, we have uncovered a graded distribution of threedorsal-pallial transcripts that in zebrafish allow us todistinguish three different areas of the dorsal pallium: ananteromedial domain, a dorsal domain, and a caudolateraldomain. The homology of these domains with known divi-sions of the mammalian isocortex will be discussed.

pcp4a is a marker of reticulospinal neuronsthroughout development

The first groups of neurons to express pcp4a are theprecursors of the reticulospinal neurons that develop inthe laterodorsal region of the hindbrain, in r5 and r6,between 8 and 26 hpf. The RS neurons are among the firstneuronal units to become active in the zebrafish larvae,and they mediate the escape response (Liu and Fetcho,1999), the first coordinated neuronal reflex. There are veryfew molecular markers that allow us to identify these cellsprior to the extension of their axons into the spinal cord,val being the best characterized, but it labels only theMauthner neurons and another pair of RS neurons in r5(Moens et al., 1996). Traditionally the RS neurons areidentified through retrograde labeling. Thus the first in-dication of the origin and location of these neurons relieson our ability to label them retrogradely. Detailed studiesof their number, location, and projections between 20 hpfand 5 dpf have been carried out (Mendelson, 1986a; Men-delson, 1986b; Metcalfe et al., 1986).

With the identification of pcp4a as an early marker ofreticulospinal neurons and the study of its expression inRS neurons at various stages, new insights into the originof these cells were obtained. It appears that these neuronsare born/specified in a more lateral position than wherethey will eventually reside. The lateral origin of these cellswas suggested by Mendelson (1986b) in his time coursestudy using HRP to label RS neurons retrogradely as earlyas 24 hpf. A dorsolateral origin of hindbrain projectionneurons, followed by a ventral migration, similar to thatseemingly undertaken by zebrafish RS neurons, has beendescribed in chick (Clarke et al., 1998).

In addition, from the analysis of pcp4a expression, itappears that some RS neurons are present at early stagesas clusters of pcp4a-expressing cells, which subsequently

Fig. 8 (Overleaf). pcp4a expression in the hindbrain. A–C: Whole-mount in situ staining of the hindbrain region of 5-dpf zebrafishlarvae. A: Backfilling of reticulospinal neurons with fluorescein dex-tran. M, Mauthner neurons. B,C: pcp4a in situ hybridization depictedat two different planes of focus. White arrow indicates the position ofthe Mauthner neurons, which are not stained with the pcp4a probe.Compare with J. Single asterisk indicates the Mid3cm cell, and doubleasterisks indicate the Cad reticulospinal neurons, all expressingpcp4a. D–F: Frozen sagittal sections through the brainstem of2-week-old zebrafish at the level of the lobus (L) VII and X. Boxedregions are enlarged in E and F. E: Arrow points to the descendingaxon of a reticulospinal neuron enriched with pcp4a transcripts.F: More neurites expressing pcp4a transcripts in the upper spinalcord (arrows). G,H: Transverse sections through the hindbrain of5-dpf (G) and 1-month-old zebrafish (H) showing the high levels ofpcp4a expression in the Mid3cm cells (asterisk). I: High levels ofpcp4a transcripts also in the Cad group of reticulospinal neurons(double asterisk) at 2 weeks. J–L: Backfilling and in situ hybridiza-tion for pcp4a at 5 dpf, showing no pcp4a expression in the Mauthnerneurons (M; J), double staining in the Mid3cm cells (K, asterisk) andpartial backfilling of Cad neurons (L, double asterisk), some of whichexpress pcp4a (arrow). For abbreviations, see list. Scale bars � 50 �min A–L.

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Figure 9.

delaminate in single neurons that acquire different posi-tions and shape within the hindbrain. Whether this isreally what happens requires time lapse analysis andawaits the generation of a transgenic line in which the RSneurons are marked by GFP expression from their firstappearance. The pattern of location, shape, and projectionof the RS neurons is invariant and is acquired in a rela-tively short time, thus suggesting that the mechanismsthat regulate their specification, number, migration, andprojections are very robust. Studies aimed at clarifyingthe inductive signals in the hindbrain have uncovered arole for fgf signaling secreted from rhombomere 1 and 4 inpatterning the adjacent rhombomeres and in controllingthe number and identity of the RS neurons (Maves et al.,2002). In addition, a hox gene code controls the identity ofneurons developing in the different rhombomeres, includ-ing the RS neurons (Moens et al., 1996; Prince et al.,1998). The ability to identify the precursors of RS neuronsby their expression of pcp4a at earlier stages than withany other methods and throughout their life allows us toaddress questions of their specification and migration. Italso suggests that a transgenic line expressing GFP underthe control of pcp4a regulatory elements would provide anideal tool to study the development of RS neurons in liveembryos and larvae.

pcp4a expression in neuronal functionalsystems

The expression of pcp4a in different regions of the de-veloping zebrafish brain prompted us to examine whetherthese different regions and nuclei shared similar proper-ties or were part of the same sensory or motor circuitries.For example, the expression of pcp4a can be detected allalong the relay nuclei of the gustatory system, which hasbeen studied extensively by several authors in other te-leosts (Rink and Wullimann, 1998; Ahrens and Wulli-mann, 2002; Folgueira et al., 2003). The gustatory inputsare collected by the facial, glossopharyngeal, and vagalsensory roots and are conveyed to the first sensory relayneurons in the facial and vagal lobes (which expresspcp4a). Both extra-oral gustatory input (via facial sensorynerve) and intra-oral (via glossopharyngeal and vagalnerves) gustatory stimuli converge on the secondary gus-tatory nucleus in the rhombencephalon (which expressespcp4a). Then gustatory inputs are received by the tertiarygustatory nuclei in the diencephalon. These include theTGN, the TLA, the SG, and the inferior lobe. The mam-

millary body or corpus mammillare (CM) is the mostprominent paired nucleus in the inferior lobe of the te-leostean hypothalamus and expresses pcp4a at very highlevels, as do the TGN, TLA, and SG. Also, the mammillarybodies are hypothesized to relay gustatory information tomultisensory areas (Rink and Wullimann, 1998).

All these areas project to (and are interconnected with)the area dorsalis pars medialis (Dm) of the telencephalon(Rink and Wullimann, 1998; Sawai et al., 2000; Folgueiraet al., 2003; Wullimann and Mueller, 2004), which ex-presses pcp4a. Thus the expression of pcp4a is a hallmarkof the nuclei related to the gustatory system in zebrafish,which use taste discrimination not only for feeding pur-poses but also to orient and navigate toward a food source(Bardach et al., 1967; Kanwal and Finger, 1992). pcp4aexpression seems to be associated with some, but not all,relay nuclei of the somatic sensory system: for example. itis expressed in some primary sensory neurons, startingfrom early stages (Rohon-Beard cells) through adulthood(neurons in spinal sensory ganglia and trigeminal gan-glion). Many interneurons in the spinal cord expresspcp4a, probably including both inhibitory (CoLA) and ex-citatory (CiD, McoD, UcoD; Higashjijma et al., 2004) in-terneurons, which modulate the activity of segmental mo-tor neurons in response to local or general sensorystimulation.

Similarly, the visual sensory system also expressespcp4a in several relay stations, starting from the GCL inthe retina, the optic tectum, the preglomerular complex,and the area dorsalis pars lateralis (Dl), which is known toprocess visual stimuli (Nieuwenhuys et al., 1998). Becausenot all the connections and relay centers of the visualsystem in teleosts have been identified, the expression ofpcp4a may direct researchers to test whether pcp4a-positive areas may be involved with translating visual orother sensory stimuli, given its preferential expression insensory-related structures. Expression of pcp4a is not onlyconfined to the sensory system; many neurons belongingto the motor system, either as effectors (reticulospinalneurons) or as modulators (cerebellar neurons, neurons inthe area ventralis [V] of the telencephalon) also expresspcp4a. Although we do not know the functions of thispeptide in sensory and motor neurons, it is highly possiblethat they are related to its ability to bind calcium-freecalmodulin and to modulate synaptic activity.

Differential expression of pcp4a intelencephalic divisions

The homology of the divisions of the teleostean dorsaltelencephalon with those of the mammalian pallium isstill debated (Wullimann and Mueller, 2004; Mueller etal., 2004). This is due to differences in morphogenesis(eversion in teleosts versus evagination in mammals) andto differences in function (most of the teleostean dorsaltelencephalic areas are involved in multimodal sensoryprocessing, whereas several mammalian pallial areas areselectively specialized in motor or sensory [visual, audi-tory, olfactory, somatosensory] functions, in addition toassociative functions). Despite these difficulties, expres-sion data may help to find homologies in the patterningevents that gave raise to these two different structuresduring ontogeny. With a more refined analysis, in thefuture it will be possible to discover which molecules arequalitatively or quantitatively responsible for such a big

Fig. 9 (Overleaf). pcp4a expression in the spinal cord. Transverse(A,C,F–H) and sagittal (B,D,E) sections through the spinal cord of1-month (A–D) and 5-dpf (E–H) zebrafish stained for pcp4a (A,B,E–G), reelin (reln, C,D) and backfilled with biotin dextran plus pcp4a insitu (F–H). A,C: Adjacent sections; arrows point to dorsal root gangliacontaining neurons expressing pcp4a (A) but not reelin (C). Neurons ofthe intermediate lateral column express both pcp4a and reelin (ar-rowheads in A,C). E: Large cells throughout the spinal cord expresspcp4a. F–H: Most of these cells can be labeled by intraspinal injec-tions of biotin-dextran. Arrowheads point to double labeled cells;arrows in F point to backfilled cells that do not express pcp4a. Aster-isk in H points to a pcp4a� cell, not backfilled, unlike its conterpartneuron (arrowhead). Inset: Diagram illustrating the distribution ofspinal cord interneurons according to Hale et al., 2001. Scale bars �50 �m in A–H. [Color figure can be viewed in the online issue, whichis available at www.interscience.wiley.com.]

784 M. MIONE ET AL.

Figure 10.

difference in the final organization of the dorsal pallialareas in these two taxa.

Based on the observation that pcp4a expression seemsto be enriched in sensory relay nuclei, including telence-phalic areas that are hypothesized on the basis of hod-ological approaches to receive sensory inputs (Nieuwen-huys, 1998), we investigated whether the expression ofpcp4a alone or in combination with other markers couldprovide information on the molecular differences betweendorsal pallial areas of the late larval zebrafish telenceph-alon.

A graded expression of different genes (EphA7, Id2,tbr1, and others: Rubenstein et al., 1999; Emx2, Pax6,Cad6, and Cad8: Bishop et., 2000) characterizes differ-ent regions of the mammalian isocortex during develop-ment. It has been suggested that the combinatorialexpression of these genes marks functionally distinctareas of the mammalian isocortex, thus allowing one todistinguish, primarily, motor versus sensory cortices,but also visual, auditory, and somatosensory corticalareas. A complication in applying this approach to othervertebrates is that most of the molecular markers usedin mouse are also expressed outside the isocortex ordorsal pallium, in other areas of the dorsal telencepha-lon, namely, in the medial, lateral, and ventral pallium(Puelles et al., 2000). As the localization of these dorsaltelencephalic divisions in teleosts is still a matter ofdebate, and the distribution of the above transcripts inthe zebrafish larval telencephalon is largely unknown,the use of these markers is, at the moment, problematic.Therefore we decided to compare the localization ofpcp4a transcripts in different areas of the developingzebrafish dorsal pallium with that of two other dorsaltelencephalic genes that we have previously character-ized in details (tbr1, Mione et al., 2001; reln, Costagli etal., 2002). These genes have important attributes re-lated to the present study: the zebrafish olfactory sys-tem, including the area dorsalis pars posterior (Dp),which is known to receive olfactory projections (Nieu-whuys, 1998), is totally devoid of reelin expression(Costagli et al., 2002) but is enriched for tbr1 transcript-

s,as in all vertebrates (Bulfone et al., 1995; Mione et al.,2001; Brox et al., 2004). Thus, comparing tbr1 and relnexpression should allow us to identify the derivative ofthe lateral pallium in zebrafish.

Therefore, to analyze the expression of pcp4a in thedorsal telencephalon of developing zebrafish, we comparedit with that of reelin and tbr1. The results are encourag-ing, in the sense that they suggest an areal distribution ofthese three pallial-specific transcripts throughout thearea dorsalis (D) of the larval zebrafish.

It is noteworthy that other two genes, pax6 and emx2,whose transcripts show an areal distribution in thedeveloping mouse dorsal pallium and have been provedto have a causative role in the arealization of the dorsaltelencephalon in mammals (Bishop et al., 2000), are notexpressed with the same areal distribution in the dorsalpallium of 5-dpf zebrafish (see supplementary Fig. 1S).Of the two pax6 paralogues 6.1 and 6.2, only pax6.1 isexpressed in the zebrafish telencephalon at 5 dpf, in anintermediate domain between the dorsal and the ven-tral telencephalic nuclei (Wullimann and Rink, 2001;present study), and emx2 is expressed predominantly inthe olfactory bulb region, diencephalic nuclei, and hy-pothalamus (present study), in contrast to its wide ex-pression throughout the telencephalon at 24 hpf (Moritaet al., 1995; Kawahara and Dawid, 2002), and thereforeit is possible that their role in the late compartmental-ization of the dorsal pallium is not conserved through-out evolution. Emx3 mantains a strong telencephalicexpression at 5 dpf, mainly in the olfactory bulb, rostralDm and Dd, but the absence of this gene in highervertebrates (Derobert et al., 2002; Kawahara and Da-wid, 2002) makes it difficult to use its expression forcomparative purposes.

Thus, the identification of several molecularly distinctdomains of the dorsal telencephalon of larval zebrafishsuggests that rostrocaudal and mediolateral specializa-tions of the dorsal pallium are present in all vertebratespecies even if they are marked by different combinationsof regulatory genes.

ACKNOWLEDGMENTS

We thank H. Okamoto and C. Nusslein-Volhard for thegift of the transgenic lines. We are particularly grateful toSteve Wilson for advice during the course of this work andfor critically reading this article. We thank all the person-nel of the UCL Zebrafish Facility, the zebrafish group atUCL, and the UCL Confocal Unit.

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787pcp4a IN ZEBRAFISH NEURONS